An ability to control or regulate the expression of a cloned homologous or heterologous gene in a host cell is a central requirement of recombinant DNA technology. Recombinant molecules capable of mediating the expression of such genes and methods for employing such molecules are described by Cohen, et al. (U.S. Pat. Nos. 4,237,224 and 4,468,464).
Although the continual expression of a cloned gene may often be acceptable, it is frequently highly desirable to modulate the expression of a cloned gene. Several methods are currently known for achieving such regulation of gene expression.
One method for regulating gene expression employs plasmid vectors whose copy number can be amplified by increasing the temperature at which the expressing host cell is cultured. Such vectors are described, for example, by Uhlin, et al. (U.S. Pat. Nos. 4,495,287 and 4,499,189).
It is, however frequently more desirable to control gene expression through the use of highly efficient promoter regions which are capable of enabling high level expression of a cloned gene. Such promoter regions may be either constitutive, or, more preferably, regulatable (i.e. promoter regions whose capacity to mediate gene transcription can be regulated by external genes, or culture conditions). Examples of constitutive promoters include the int promoter of bacteriophage .lambda., the bla promoter of the .beta.-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage .lambda. (P.sub.L and P.sub.R), the trp, recA, lacZ, lacI, and gal promoters of E. coli, the .alpha.-amylase (Ulmanen, I., et al.. J. Bacteriol. 162:176-182 (1985)) and the .theta.-28-specific promoters of B. subtilis (Gilman, M.Z., et al., Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Bioloqv of the Bacilli, Academic Press, Inc., N.Y. (1982)), and Streotomyces promoters (Ward, J.M., et al., Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev Genet. 18:415-442 (1984)).
Examples of eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C., et al., Nature (London) 290:304-310 (1981)); and the yeast gal4 gene promoter (Johnston, S.A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, Pa., et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).
Although the above-described regulatable promoters are capable of mediating gene expression in response to changes in the conditions at which recipient cells are cultured, such regulation often requires changing the temperature of the culture media, or the introduction of small metabolites or compounds into the culture media. Such requirements are often costly, and, hence unsuitable for the large scale production of a recombinant product. Moreover, such requirement are often inefficient for large scale fermentation and production. Hence, a need exists for a method for regulating gene expression which is amenable to both small and large scale fermentations, and is both economical and efficient.